This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

Paucity of male-biased genes on the Drosophila X chromosome is a well-established phenomenon, thought to be specifically linked to
the role of these genes in reproduction and/or their expression in the meiotic male
germline. In particular, meiotic sex chromosome inactivation (MSCI) has been widely
considered a driving force behind depletion of spermatocyte-biased X-linked genes in Drosophila by analogy with mammals, even though the existence of global MCSI in Drosophila has not been proven.

Results

Microarray-based study and qRT-PCR analyses show that the dynamics of gene expression
during testis development are very similar between X-linked and autosomal genes, with both showing transcriptional activation concomitant
with meiosis. However, the genes showing at least ten-fold expression bias toward
testis are significantly underrepresented on the X chromosome. Intriguingly, the genes with similar expression bias toward tissues other
than testis, even those not apparently associated with reproduction, are also strongly
underrepresented on the X. Bioinformatics analysis shows that while tissue-specific genes often bind silencing-associated
factors in embryonic and cultured cells, this trend is less prominent for the X-linked genes.

Conclusions

Our data show that the global meiotic inactivation of the X chromosome does not occur in Drosophila. Paucity of testis-biased genes on the X appears not to be linked to reproduction or germline-specific events, but rather reflects
a general underrepresentation of tissue-biased genes on this chromosome. Our analyses
suggest that the activation/repression switch mechanisms that probably orchestrate
the highly-biased expression of tissue-specific genes are generally not efficient
on the X chromosome. This effect, probably caused by dosage compensation counteracting repression
of the X-linked genes, may be the cause of the exodus of highly tissue-biased genes to the
autosomes.

Background

Traffic of the newly-created testis-specific genes from the X chromosome to autosomes via retroposition is a major trend conserved between different taxa [1-3]. In mammals, it is believed to be an adaptation to the evolutionarily 'young' meiotic
sex chromosome inactivation (MSCI), so that the newly-duplicated autosomal genes 'rescue'
the silencing of the parental X-linked genes in the male germline [4]. This process would lead to the loss of the X-linked genes that function specifically in the MSCI-affected male germline cells,
namely spermatocytes. Indeed, spermatocyte-specific genes are severely underrepresented
on the X chromosome in mice, illustrating the proposed role of MSCI in genome evolution. In
contrast, the male-specific genes expressed at either earlier or later stages of spermatogenesis
(that is in spermatogonia or in round spermatids) are over-represented on the mouse
X chromosome [5,6], consistent with the vigorous selection for male-beneficial X-linked genes proposed previously [7].

Reports on the existence of MSCI as a mechanism of global inactivation of the X-linked genes in spermatocytes of Drosophila are not conclusive. In flies, similar to mammals, spermatocytes rapidly proceed through
the S-phase and enter extended prophase I of meiosis, which lasts several days (reviewed
in [8]). The premature cytological condensation of the X chromosome in primary spermatocytes and the dominant male sterility frequently caused
by X-autosomal translocations were originally put forward as indicators of MSCI in Drosophila [9]. The overall paucity of X-linked testis-specific genes and the 'exodus' of the newly-duplicated testis-specific
genes from the X chromosome via retroposition have been reported in flies and interpreted as additional evidence of
MSCI [1,10-14]. However, a recent study indicates that retroposing genes avoid the X regardless of their expression pattern, and probably owing to their intrinsic integration
preferences [15]. The finding that the X-linked transgenes driven by a testis-specific ocn promoter fail to show active expression [16] further supported the MSCI model, even though the 'sampling' of chromosomes by the
transgene insertions was not very comprehensive. Finally, microarray analyses of dissected
testes showed lower X-linked gene expression in the sample enriched with spermatocytes as compared to samples
that included earlier or later developmental stages [17]. However, this finding could have been affected by differences in the somatic cell
content between different regions of testis because somatic cells are underrepresented
in the spermatocyte-enriched sample; therefore, the genes expressed in somatic cells,
but not in germline, could be perceived as inactivated in spermatocytes. On the other
hand, no significant dearth of X-linked transcription has been detected in another genome-wide analysis of gene expression
in gonads, arguing against global inactivation of the X chromosome in the meiotic germline [18]. However, active gene expression in spermatogonia and somatic testis cells [19] present in the whole adult testes could in principle 'mask' the putative gene silencing
in meiotic cells in this study.

In order to understand better the genome evolution and structure, and to obtain further
insight into the tissue-specific gene expression, it is important to determine whether
the paucity of male-biased X-linked genes in flies is due to MSCI or other novel, yet unknown mechanism(s). To
determine whether the expression of X-linked genes changes concomitant with meiosis, we performed a study of the testis
transcriptome in larval development, tracing the maturation of the first wave of spermatocytes
through the third instar and up to the meiotic divisions at the beginning of pupation
[20]. In this model, the 'load' of spermatogonia and associated somatic cells is similar
between the developmental stages; therefore the differences in gene expression are
attributable to an increase in the number of spermatocytes and to their maturation.
While the expression of testis-specific X-linked genes steadily increased during spermatocyte maturation as expected, global
gene expression on the X chromosome did not significantly differ from the autosomes throughout the entire period
- both results being consistent with the lack of global MSCI. Further, we found that
the paucity of testis-biased genes is not a unique example and that other tissue-biased
genes are also underrepresented on the X chromosome, indicative of this trend not being limited to the reproduction-related
genes. Our analyses suggest that the paucity of X-linked tissue-specific genes may be driven by the general inefficiency of stringent
tissue-specific gene regulation on the X chromosome.

Results and Discussion

X-linked and autosomal testis-biased genes show similar longitudinal profiles of expression
in testis development

MSCI should lead to a paucity of the spermatocyte-specific genes on the X chromosome, as has been shown in mammals [5]. In Drosophila, the vast majority of the testis-specific genes analyzed to date are activated in
primary spermatocytes, that is in the meiotic prophase I [21]. To determine whether expression profiles of X-linked genes deviate from this trend, we quantitatively analyzed the transcription
of eight previously characterized testis-biased X-linked genes in larval testis development, using real-time RT-PCR. In parallel, 18
autosomal testis-biased genes were analyzed as a reference group (Table 1) [22-25].

Table 1. The X-linked and autosomal spermatocyte-specific genes analyzed for their temporal expression
during testis development

Testes were collected daily from day four (second instar) through day seven (Figure
1, dL), at which point third-instar larvae started to pupate (Figure 1, dP). Since the first wave of germline differentiation reaches meiotic divisions
approximately at the onset of pupation [19] and spermatocytes become the predominant cell type in testes of third instar larvae
[26], this analysis traced changes in gene expression associated with the accumulation
and maturation of spermatocytes. Accordingly, transcripts of known spermatocyte-specific
genes, both autosomal and X-linked, showed a steady increase throughout testis development in our series of samples
(dotted lines on Figure 1A, B). Other analyzed genes, including those located on the X chromosome, showed the same pattern indicating that their activation occurs in the
meiotic germline. The pooled data also show a striking similarity of the expression
patterns between the groups of X-linked and autosomal genes (Figure 1C). We further sought to determine whether the relatively small selection of X-linked genes for our study could have influenced our conclusion that these genes
are usually activated concomitant with meiosis. To assess a larger sample, we analyzed
published in situ hybridization data for 501 X-linked genes [21] and found that 474 of them (94%) are induced in primary spermatocytes. Cumulatively,
the above data obtained by different approaches convincingly show that numerous X-linked genes are activated in the male meiotic germline. It is not clear then why
the transgenes driven by promoter of the testis-specific ocn gene could not show efficient expression when integrated into the X chromosome [16], given that ocn shows the same expression profile as endogenous X-linked testis-biased genes (compare the solid line on Figure 1B for ocn to the profiles in Figure 1A). However, regardless of the mechanism(s) of silencing, the ocn transgenes [16] do not appear representative of the X-linked genes that are commonly expressed in spermatocytes.

Figure 1.X-linked and the autosomal testis-biased genes show similar patterns of activation
in testis development. (A) X-linked genes; the profile for the known spermatocyte-specific gene Sdic is outlined as a black dotted line. Other genes are CG15450, CG1314, CG1338, CG15711, CG15452, CG11227 and CG1324 (gray lines). (B) autosomal genes; the profiles for the known spermatocyte-specific
genes fzo, dj, and β(2)Tubulin are outlined as black dotted lines. The profile for the gene ocn which is the source of promoter in the transgene based study of Hense et al. [15] is outlined as a black solid line. Other genes are CG6262, CG3483, CG7813, CG3492, CG15874, CG3494, CG16837, CG4439, CG4750, CG15873, CG15925, CG7848, CG15710, Eyc, Mst35Ba, and Mst35Bb (gray lines). (C) Box plot analysis of gene expression data (A, B) shows no significant
differences in the expression patterns between the X-linked (orange) and the autosomal (blue) gene sets. For each gene, expression level
in pupae served as the reference.

Global gene expression on the X chromosome does not significantly change during testis development

To further support our findings by extending them to the majority of X-linked genes, we analyzed the global trends of X-linked gene expression during testis development using gene microarrays. To improve
the resolution of the analysis, we cultured larvae at a lower temperature (18°C) and
collected testes from feeding larvae at fourth, fifth, sixth and seventh days of development
(Figure 2 dLf), and from wandering larvae at days seven and ten, just before the onset of pupation
at days 11 to 12 (Figure 2, dLw). Throughout larval testis development, we did not find appreciable differences in
expression between the X chromosome and autosomes (Figure 2, orange versus blue bars) or any substantial reduction in X-linked gene expression (Figure 2, orange bars), strengthening the hypothesis that the X chromosome is not affected by global MSCI in Drosophila, contrary to mammalian spermatogenesis.

Figure 2.Lack of global X-chromosome inactivation in developing testes. Expression of X-linked (orange) and autosomal (blue) genes was measured as signal intensity of corresponding
microarray probes, after normalization. cDNAs hybridized to the microarrays were isolated
from testes of either feeding (f) or wandering (w) larvae grown for the indicated
number of days at 18°C. The analysis traces the first wave of germline differentiation;
pupation indicative of the meiotic divisions occurred at day 11.

Weak testis bias of expression shown by X-linked genes

Although we did observe numerous X-linked genes increasing their expression during testis development, previous reports
showed a paucity of male-biased genes on the X [12-14]. To define the relationship between these two gene categories, we analyzed expression
bias of the genes up-regulated in developing testes. Using published gene expression
data [27], we compared microarray signals observed with adult testis-derived RNA to the signals
obtained with RNA from a variety of somatic sources including accessory glands, brain,
head, thoracicoabdominal ganglion, crop, midgut, hindgut, malpigian tubules of adults
and larvae, ovary, salivary glands, and carcass. Conservatively, the minimal testis-to-somatic
tissue signal ratio across the entire panel of analyzed samples was defined as a measure
of bias of gene expression toward testis. This analysis identified about 50% of the
autosomal genes up-regulated in testis development as highly testis-biased (minimal
testis::somatic tissue signal ratio ≥10) (Figure 3, blue bars). Strikingly, highly-biased genes constitute less than one-quarter of
the X-linked genes, and this difference from the autosomal genes is highly significant
(χ2 test, P = 3 × 10-10) (Figure 3, orange bars). Apparently, it is the cohort of highly testis-biased genes that is
depleted in the pool of the X-linked genes up-regulated in testis development. As a result, on average the X-linked genes show a weaker testis bias of expression than their autosomal counterparts.

Figure 3.Paucity of X-linked genes with high expression bias toward testis. The testis::somatic tissue ratio of microarray signal intensities, observed between
adult testis and a panel of somatic tissues [24], serves as a measure of testis bias of gene expression and is shown at bottom. Bars
indicate proportion of genes up-regulated in testis development in the four bias categories.
Orange bars, X-linked genes; blue bars, autosomal genes.

Diverse tissue-specific genes are underrepresented on the X chromosome

Our gene expression study on developing testes indicated that the paucity of testis-biased
genes on the X chromosome is not caused by the spermatocyte-specific events. We therefore hypothesized
that the underlying mechanisms may be not restricted to the male meiotic germline
and instead could operate in diverse tissues, causing broad effects on tissue-biased
expression. To test this suggestion, using the published gene expression dataset [27] we identified genes that show expression bias toward midgut, malpigian tubules, accessory
gland, salivary gland, head, and ovary, using the same bioinformatics approach as
described above for testis. We further determined whether the frequencies of the identified
genes on the X chromosome deviated from the genome averages. We observed that the majority of the
analyzed genes show the same trends that the testis-biased genes do: they are underrepresented
on the X chromosome, and the higher the expression bias the stronger the underrepresentation
(Figure 4). One important exception, however, was the ovary-biased gene set for which both
trends were reversed. This finding is consistent with previous reports on the overrepresentation
of female- and ovary-biased genes on the X [13,28], and indicates that the genes selectively expressed in the ovary are subject to unique
selective pressures, probably owing to their female-beneficial sexually-antagonistic
effects [7,29] and/or because of their peculiar regulation, as discussed below.

Figure 4.Highly tissue-biased genes show a skewed representation on the X chromosome. The ratio of microarray signal intensities observed between the tissue sample indicated
at bottom including midgut, malpigian tubule, accessory gland, salivary gland, head,
and ovary and a panel of other tissue samples [24] was used as a measure of tissue bias. The bars correspond to the frequencies of the
genes on the X chromosome normalized against the genome averages, and are shown for
the genes with at least two-fold expression bias toward indicated tissues (light gray),
for the genes with at least five-fold bias (dark gray), and for highly biased genes
with at least ten-fold bias (black).

Genes activated in testis development appear to be repressed in embryos and cultured
somatic cells

Based on our analyses, we suggest that the X chromosome provides an inferior environment for specialized genes with expression
highly biased toward particular differentiated cell types. To gain a better understanding
of the underlying mechanisms, we analyzed the binding of diverse chromatin proteins
to the X-linked and autosomal tissue-biased genes. First, we analyzed the correlations between
gene expression in testis development and binding of 27 chromatin proteins in embryonic
and cultured cells. Changes in gene expression were measured as signal fold changes
for every time point in our microarray-based analysis; the earliest analyzed stage
(four-day old larvae) served as the reference. Chromatin protein binding was assessed
as the fold enrichment in chromatin immunoprecipitation or in DNA adenine methyltransferase
identification experiments [30,31]. We have found a correlation that was 'inversed' with respect to the major roles
of the analyzed proteins: that is, gene up-regulation in testis development was positively
correlated with the binding of silencing-associated proteins in embryos and cultured
cells (Figure 5, results for proteins with insignificant correlations are not shown). Specifically,
testis-up-regulated genes showed prominent associations with histone H1, histone H3
trimethylated at K27, LamDmo, D1, and Polycomb (Pc) and Pc group proteins esc and Sce [25,32-38]. In addition, a negative correlation was apparent between genes up-regulated in testis
and the binding of gene activation-associated proteins in embryos and cultured cells
(these proteins include histone variant H3.3A, H3 trimethylated at K4, DJun, and bcd)
[39-41]. These observations imply that testis-biased expression is regulated by a two-prong
'switch' mechanism comprised of gene activation in testis (most likely, male germline)
and gene repression in other tissues and cell types.

Figure 5.'Inversed' correlations between gene regulation in testis development and chromatin
modifications in somatic cells. For each of the developmental time points indicated on the horizontal axis, correlations
were calculated between up-regulation of genes in testis relative to the earliest
time point (from microarray data) and binding of the same genes to the shown chromatin
proteins in embryos or somatic cells [29,30]. Yellow, orange, and purple colors represent proteins that are generally associated
with gene silencing, and blue and green colors represent proteins generally associated
with active gene expression.

We inquired whether the tissue-biased gene activation generally correlates with binding
of repressor proteins in the 'non-target' embryonic and cultured cells, and whether
such correlation differs between the X chromosome and autosomes. For this analysis, we calculated the relative enrichment
with chromatin proteins for the genes that show more than two-fold expression bias
toward testis, midgut, accessory gland, salivary gland, malpigian tubule, and ovary.
The tissue expression bias was determined using the same approach as described above
for the analysis of tissue-biased gene frequencies on chromosomes. Using arbitrarily-set
thresholds to define the categories of bound versus non-bound genes for each of the analyzed chromatin proteins [30,31], we determined the frequencies of bound genes in the sets of tissue-biased genes
on the X chromosome and autosomes and normalized them to the frequencies of bound genes in
the entire genome. The study included nine repressor proteins and eight activators
that showed the strongest 'inverse' correlations in previous analysis. The resulting
graphs (Figure 6) show whether the X-linked (orange bars) or autosomal (blue bars) genes bind repressors or activators
in embryos and cultured cells with a frequency different from the genome average (defined
as 1.0).

Figure 6.The autosomal and the X-linked tissue-biased genes show different patterns of chromatin modifications. The frequencies of binding targets for the proteins indicated at the bottom were
calculated among the X-linked (orange) and autosomal (blue) tissue-biased genes. The sets of genes showing
at least two-fold expression bias toward the indicated tissues (such as testis, midgut,
accessory gland, salivary gland, malpigian tubule, and ovary) were generated from
the genome-wide expression data [24]. Protein binding genes were defined using chromatin immunoprecipitation and DamID
data [29,30] with arbitrarily set thresholds. Bars show relative increase or decrease in binding
target frequency within analyzed gene sets as compared to the entire genome.

Consistent with the above correlation studies, this analysis showed that autosomal
testis-biased genes are enriched with repressors and rarely bind activators in embryos
and cultured cells. In the case of the X-linked genes this trend is reduced for the vast majority of the analyzed proteins
and even reversed for three repressors and five activators (Figure 6). Therefore, the drastic differences in expression status between germline and somatic
cells are less common for the testis-biased genes located on the X, indicating that the activation/repression 'switch' is not prominent in the regulation
of these genes. Similar observations were made for the majority of genes with an expression
bias toward other tissues, with a few exceptions including the malpigian tubule-biased
autosomal genes showing enrichment with activators, and the salivary gland-biased
X-linked genes showing strong enrichment with repressors (Figure 6). In general however, we found that, unlike their autosomal counterparts, the X-linked tissue-biased genes are not enriched with repressors and do not lack activators
in embryos and cultured cells, implying that the tissue-biased up-regulation of the
X-linked genes is usually not complemented by their repression in other, 'non-target'
cell types. In this respect, ovary-biased genes may provide an example of tissue-biased
genes that apparently do not rely on the activation/repression mechanism, regardless
of their chromosomal location, because, unlike other tissue-biased genes, they are
strongly associated with activators and do not bind repressors in embryonic and cultured
cells. This unique feature may facilitate accumulation of the ovary-biased genes on
the X chromosome.

Conclusions

Our findings presented in this study strongly suggest that global inactivation of
the X chromosome does not occur in the Drosophila meiotic male germline, and therefore other factors and mechanisms should be invoked
to explain the peculiarities of X-linked gene expression. A study of gene expression in testis development using microarrays
and qRT-PCR, combined with analysis of published in situ hybridization data convincingly show that the majority of X-linked genes do not undergo silencing concomitant with meiosis; in fact, many of
them are activated in the meiotic germline cells. In agreement with the analysis of
sex-biased expression in several Drosophila species [18], we conclude that the possible appearance of X-chromosome underexpression in male gonads results from the paucity of X-linked testis-biased genes rather than from chromosome-wide gene silencing. Why the
X chromosome behaves in meiotic cells in Drosophila unlike in mammals is not clear; perhaps the yet unidentified mechanisms that cause
overexpression of X-linked genes in the Drosophila germline [18] counteract global silencing of the X chromosome.

Our findings could be consistent with the sexual antagonism driving X inactivation (SAXI) model [10], in which the pressure of sexual selection drives the 'exodus' of male germline-specific
genes from the X chromosome causing its inactivation in meiotic cells, with the one exception that
no true global X inactivation is seen, just the lack of testis-biased genes on the X. However, a recent study indicates that retroposing genes with diverse expression
biases all show an affinity toward autosomes [15]; therefore the apparent 'exodus' of male-biased genes from the X, previously considered as the major indirect evidence for MSCI in flies [1], is not necessarily driven by sexual selection. Earlier studies did show that not
only testis-biased, but also somatic male-biased genes are underrepresented on the
X [42], but the possible role of these genes in sexual selection could still be invoked
with certain ease owing to their sex bias. Here, we showed that the genes with strong
expression bias toward tissues that are not directly related to reproduction, such
as salivary glands, midgut, and malpigian tubules, also show paucity on the X chromosome. Taking into account the recent report that somatic gene expression may
be regulated by gonads [43], we inquired whether the gonad-dependent genes appreciably contributed to our tissue-biased
gene sets. The heaviest representation of gonad-dependent genes [43] was found, not surprisingly, in the testis-biased and ovary-biased gene sets but
in both cases it was less than 4%, and less than 2% of the genes preferentially expressed
in salivary glands, midgut, or malpigian tubules appear to be gonad-dependent. Thus,
the paucity of tissue-biased genes, and probably the underlying 'exodus' of such genes
from the X chromosome, involves genes that have no apparent specific role in reproduction.

A possible clue to the reasons why tissue-biased genes are so scarce on the X is provided by the finding that the genes with the highest bias are affected much
more than the genes with modest expression differences (Figure 4B). Based on the analysis of chromatin protein binding to the biased genes, we hypothesize
that the high expression bias results from the combined effect of gene activation
in the 'target' cells and repression in other cell types. Our analysis indicates that
such a mechanism does not operate frequently on the X-linked tissue-biased genes. This finding presents 'the chicken or the egg' dilemma
because we cannot discern whether the weak tissue bias of expression results from
not using the activation/repression 'switch' mechanism, or vice versa. Nevertheless, it is possible that the inefficiency of this 'switch' on the X chromosome significantly affects genome evolution. The importance of this influence
is illustrated by the unique example of the ovary-biased genes that apparently do
not rely on the activation/repression mechanism (Figure 6) and strongly accumulate on the X chromosome (Figure 4) in perfect agreement with the female-beneficial selection model [7,29].

Of the two components of the activation/repression switch, the repression may be vulnerable
to the X chromosome-specific mechanism of dosage compensation that broadly and ubiquitously
activates X-linked genes in somatic cells [44]. To test this suggestion, we analyzed binding of the testis-up-regulated genes with
the key dosage compensation component msl-2 using published datasets [45,46] expecting to find that the highly testis-biased genes do not bind msl-2. We found
no correlation between testis bias of expression and msl-2 binding. However, dosage
compensation machinery can regulate genes at a distance from the primary binding sites
[47] and a recent study did show that the genes with the highest male bias of expression
tend to localize outside the compensated regions [48]. We propose, therefore, that the H4-K16 acetylation of the X chromosome by the dosage compensation complex interferes with highly tissue-biased
gene expression by counteracting repression in the 'non-target' cell types. This may
present a significant problem if the gene product is toxic to diverse cells (which
for example could be expected for many sperm differentiation genes), and could result
in a strong selective pressure toward relocation of the highly tissue-biased genes
to the autosomes.

Methods

Fly culture and dissection

Wild type (Oregon) larvae were grown on yeast-molasses media at 18°C. At the specified
time points larvae were collected, and testes harvested using manual dissection. Further
analyses utilized samples pooled from at least 30 larvae.

Microarray probes

Total RNA was isolated from testes with Trizol reagent (Invitrogen), and further purified
with the RNeasy kit (Qiagen, Hilden, North Rhine-Westphalia, Germany). Poly(A)+ RNA
was selectively amplified from 0.5 to 1 μg total RNA samples with the BD SMART cDNA
Synthesis kit and the BD Advantage 2 PCR Enzyme System (Clontech, Mountain View, CA,
USA). Amplified cDNAs were cleaned with the Wizard SV Gel kit (Promega, Madison, WI,
USA). cDNA samples of 3.5 to 4.5 μg were labeled with the ULYSIS Alexa Fluor 546 or
Alexa Fluor 647 (Molecular Probes, Eugene, OR, USA) dyes according to the manufacturer's
protocol, and used for competitive hybridization with microarrays.

Hybridization of samples to microarrays

The Drosophila oligonucleotide microarray set (Qiagen-Operon) was printed on aminosilane-coated slides
at Tufts-New England Medical Center Expression Array Core facility (TEAC). The set
contains 14,593 70-mer oligonucleotides representing 13,664 genes which cover most
of genes in release 3.2 of the Drosophila genome (The FlyBase Consortium, 2003). The probe sequences from the Operon set were
re-mapped to Drosophila genome release 5.1 using BLAST, and probes with multiple hits or ambiguous mapping
were excluded from subsequent analysis. Additional information on the Drosophila oligonucleotide set can be found at the manufacturer's website http://www.operon.comwebcite. Details of the array design can be found at ArrayExpress (array design Operon AROS
D. melanogaster v.1.1). Labeled pairs of samples were hybridized in 1 × hybridization
buffer (GE Healthcare, Little Chalfont, Buckinghamshire, UK), 20% formamide, and 0.025%
each of Ficoll, polyvinylpyrrolidone, Na pyrophosphate, and heparin. Samples were
incubated with microarrays for 44 hours at 37°C. After hybridization slides were washed,
dried by centrifugation, and scanned on the ScanArray 4000 scanner (PerkinElmer, Waltham,
MA, USA).

Data acquisition and analysis

Fluorescence intensities of individual spots were acquired from the array images with
ImaGene software (BioDiscovery). Subsequent normalization and statistical analysis
were performed with package limma [49,50], part of the BioConductor project [51]. Normalization of the data is described in Normalization protocol associated with
ArrayExpress submission E-MEXP-1980 of the microarray experiment described in this
manuscript. At least three hybridization replicates passing quality control were performed
per experimental condition. Data were analyzed using the linear modeling approach,
and Bayes moderated t test was used to determine differential expression between each
time point and the fourth day (the earliest time point). q values for individual contrasts
represent FDR for multiple testing [52]. q < 0.1 was chosen as the threshold for differential expression unless specified
differently.

Hypergeometric analysis of the representation of differentially expressed genes on
Drosophila chromosomes was performed with the web-based tool GeneMerge (http://genemerge.cbcb.umd.edu/webcite[53]). Significance of the differences in bin content for different chromosome arms (see
Figure 4 and related text in Results section) was assessed with Fisher's exact test as implemented
in R, and correlations between the gene expression and protein binding data were calculated
with two-sided Pearson's method, also as implemented in R [54]. The significance of Fisher's and Pearson's tests was adjusted for multiple testing
according to Bonferroni method within corresponding data series.